Platelet Factor 4 (PF4) is a platelet chemokine that is synthesized during megakaryopoiesis and stored in agranules. PF4 is known to inhibit megakaryocyte development in vitro. We now show that during megakaryopoiesis, PF4 is released by megakaryocytes in amounts sufficient to affect platelet counts in mice. Further, endogenous PF4 levels affect the degree and duration of thrombocytopenia after intramedullary megakaryocyte lysis following chemotherapy-induced thrombocytopenia (CIT). Strategies that block PF4's effect ameliorate this CIT. We have also begun to define the molecular basis of this negative paracrine loop. A Low Density Lipoprotein Receptor-related Receptor appears to be the target receptor. These observations will now be pursued in three specific aims: Specific Aim 1: Define the molecular basis of the PF4 negative paracrine loop on megakaryopoiesis. These studies will define the target progenitor cell involved. The nature of the receptor will be confirmed and downstream intracellular events will be defined. The studies in this Aim should provide important insights into the molecular basis of megakaryopoiesis and may lead to novel strategies for preventing thrombocytopenia in a number of clinically relevant settings. Specific Aim 2: Characterize the in vivo effect of PF4 on megakaryopoiesis. These studies will extend observations already made on the role of PF4 content on the degree and duration of thrombocytopenia following marrow injury. We will define the interaction of the PF4 negative paracrine loop with positive cytokines (e.g., thrombopoietin). The importance of PF4 after repeated cycles of CIT or radiation therapy will be examined as will additional strategies for targeted block of this PF4 inhibitory loop. The studies in this aim should complement those in Aim 1, providing new insights into the PF4 negative paracrine loop that may be clinically relevant. Specific Aim 3: Clinical studies of the PF4 negative paracrine loop in megakaryopoiesis. Based on preliminary studies of platelet PF4 levels in the general population, we propose that ~10% of patients will be especially sensitive to chemotherapeutic marrow ablation due to platelet PF4 levels. We will analyze the severity of thrombocytopenia in patients with Acute Lymphoblastic Leukemia during delayed intensification and relate it to PF4 levels. We believe that this study will help to translate our in vitro and murine in vivo studies to clinical relevancy. This study should be an important bridge to future studies developing novel strategies for the prevention of CIT and thrombocytopenia in other settings associated with intramedullary lysis of megakaryocytes. It is becoming clear that thrombocytopenia in a number of clinical relevant models involves intramedullary megakaryocyte lysis. We believe that the studies proposed in this application will provide novel new insights to explain the degree of thrombocytopenia seen and may provide novel therapeutic approaches for its treatment.